枸杞液泡膜H^+-转运焦磷酸酶基因LbVP1克隆及表达分析

Cloning and Transcriptional Analysis of Vacuolar H^+-pyrophosphatase Gene LbVP1 from Lycium barbarum L.

液泡膜H^+-转运焦磷酸酶在调节植物生长发育和逆境胁迫应答中发挥重要作用。本研究在已构建的枸杞本地数据库中先进行VP基因电子克隆,然后结合RT-PCR方法克隆到一个枸杞液泡膜H^+-转运焦磷酸酶编码基因LbVP1,并对其序列结构、时空表达模式进行了初步分析。LbVP1开放阅读框全长2301 bp,编码766氨基酸残基。多序列比对显示该基因与拟南芥AVP基因相似性达87.9%,序列结构生物信息学预测和亚细胞定位实验表明,该基因具有液泡膜H^+-转运焦磷酸酶的保守结构域和跨膜区,可能在液泡膜发挥作用。荧光定量PCR结果显示,LbVP1在花、叶和不同发育阶段果实中存在时空表达差异,干旱胁迫处理下叶片和果实中该基因表达量与对照存在(极)显著差异(P<0.01,P <0.05)。研究结果将为阐明该基因在枸杞抗旱分子调控机制中的作用提供基础理论依据。

Vacuolar H+-pyrophosphatase plays an important role in plant growth and stress response.By taking use of in-silico cloning and RT-PCR,here we isolated a vacuolar H+-PPase cDNA(named LbVP1)from Lycium barbarum L.and analyzed the spatiotemporal expression pattern and its sequence structure.The open reading frame of LbVP1 expanded 2301 bp which encodes for 766 amino acid residues.Sequence alignment suggested a 87.9%identity of the putative LbVP1 protein in relation to Arabidopsis thaliana(L.)Heynh.VP1.This deduced protein contained conserved domains and amino acid residues with the H+-PPase proteins from other plants.By transient expression in Arabidopsis thaliana(L.)Heynh.protoplast,LbVP1 was found mainly in the vacuolar membrane.The quantitative real-time PCR reflected variations on expression of LbVP1 varied in flower,leaves and fruit of different development stages.Under drought stress,its transcriptional level of this gene in leaves and fruits was significantly different in relative to the controls.Thus,these results will provide insights for elucidating the molecular mechanism of LbVP1 in response to the drought stress.